Comments on: RNA Journal Club 7/9/09

By: Debora Marks

Debora Marks — Thu, 30 Jul 2009 19:36:36 +0000

We did indeed test the HCT116 dicer hypomorphs in this paper and we do see some saturation -like effect. This may be because these cells do have significant mature microRNA expression despite the homozygous deletion of the helicase domain of dicer. See Cummins et al., 2005 esp Figure 5.
(If you examine Fig 5B you will see that whilst many microRNAS are indeed down-regulated in the hypermorph, some clearly aren’t.)
On the other hand the absolute amounts of the expression changes should be lower, this should be clear in the original data. I will check
.

By: Graeme Doran

Graeme Doran — Sun, 26 Jul 2009 21:25:50 +0000

Thanks, I enjoyed the paper.

I don’t know anybody that has a confident figure for AGO protein numbers yet, not sure that the antibodies available are good/comprehensive enough. There is also the issue of how different si/mi are distributed amongst the family members.

One comment that came out of the discussion of the paper that I forgot to mention – it would be nice if the de-repression signal was absent/reduced in when using siRNA in DICER knockout cells (and maybe even in the hypomoph HCT116 line), on the assumption that there is more free AGO# in these cells. Not sure whether there is array data to that end yet though.

By: Debora Marks

Debora Marks — Sat, 25 Jul 2009 12:57:41 +0000

Thanks for this interesting summary of our work. Answering a few of the questions;
Yes, we did also look at sites without conservation and still got a clear signal ( data unpublished) Happy to share this if anyone wants it. The signal was stronger using conservation and worked for single sites . Much stronger for multiple sites vs single when not using conservation for obvious reasons. Remember that all, even ‘gold-standard’ target predictions are only getting 1:8 targets correct maximally ( false positives), so its pretty amazing we got a signal at all.
As for the dose-titration data. maybe there’s a misunderstanding or we weren’t clear enough in the paper. We used unconserved sites so counted all those with 2 or more ( since so many genes have one site unconserved for cellular microRNAs) and we wanted to use the siRNA data. As you know from other work ( eg Jackson 2009) siRNA sites in target mRNAs maybe less likely to be conserved than microRNA sites. We would love to have more dose data and are in the process of analyzing some new data released 2009.
Your miR-21/SMAD pathway point is interesting. We touch on it in the paper by intimating that there may be ‘physiological’ conditions where ‘saturation/competition’ is used by the cell to down-regulate the ‘older’ endogenous microRNAs thereby up-regulate their targets. If Argonaute was limiting physiologically, this would be a cute way for the cell to switch regulatory programs before having to degrade all the ‘older’ microRNAs. That is, by sponging up the AGO. You can imagine this is how viral microRNAs might work too.
One line of evidence in support of AGO being limiting in the cell even before adding ectopic mi/siRNAs, is that we see a signal when microRNAs are inhibited. But we need more data on that.
Screams out for some experiments and a model, and there’s no substitute for knowing the real numbers.
Do we know the concentration of AGO2 ( for example) in any cell type?